Biofilm infections on body surfaces or medical devices represent a serious therapeutic challenge since organisms are recalcitrant to conventional antibiotics. Therefore, an urgent need exists to approach biofilm biology from unique perspectives and employ collaborative approaches to facilitate the identification of novel treatment paradigms. The current consensus is that biofilms evade the host immune response; however, the available reports have focused on classical anti-bacterial mechanisms operative during planktonic growth and have neglected to examine the possibility that biofilms induce an immune deviation towards alternative anti-inflammatory pathways. To date, only one report has examined the immune response to S. aureus biofilm and further in-depth studies are warranted given the propensity of these infections to disseminate and colonize other sites in the body. The hypothesis of this proposal is that S. aureus biofilm skews the host innate immune response from a classical pro-inflammatory bactericidal phenotype towards an anti-inflammatory, pro-fibrotic response to favor bacterial persistence. We will focus on the functional importance of key bacterial recognition molecules (Toll-like receptor 2 (TLR2) and TLR9) as well as anti-microbial mediators (INOS) by studying the pathogenesis of S. aureus biofilm using a foreign body infection model in mice deficient for these various molecules. We will also investigate the effects of purified neutrophils, macrophages, and dendritic cells on biofilm survival and whether these immune cells induce changes in biofilm gene expression by transcriptional profiling. In addition, we will employ in vivo bioluminescence imaging (IVIS) to monitor the immune response throughout biofilm development utilizing reporter mouse strains engineered to express luciferase under the control of promoters pivotal in host immunity to gram-positive bacteria (i.e. INOS, TLR2, and NF-KB). Finally, we will examine the host immune response to S. aureus murein hydrolase {cidA and IrgAB). nuclease {nuc), and nitric oxide reductase {nor) mutants from Projects 1 and 3 of this PPG in the mouse foreign body infection model. To address these objectives, the following specific aims will be investigated: 1) compare host innate immune responses to S. aureus biofilm versus planktonic infection; 2) define the role of extracellular bacterial DNA (eDNA) and peptidoglycan (PGN) in modulating host innate immunity to S. aureus biofilm; and 3) examine the immune mechanisms leading to fibrotic encapsulation of S. aureus biofilms. The studies described in this proposal will employ a comprehensive approach to investigate the innate immune response to biofilm both in vitro and in vivo taking advantage of our experience in studying innate immunity. Understanding the cross-talk between S. aureus biofilm and innate immune cells may identify novel candidates to target for drug discovery to disrupt biofilm growth.